Biology subjects

Fang, H.

Publications and source records attributed to Fang, H..

At least 55 records · Page 3Linked to original sources

bioRxiv2023

CTCF-mediated insulation and chromatin environment modulate Car5b escape from X inactivation

BackgroundThe number and escape levels of genes that escape X chromosome inactivation (XCI) in female somatic cells vary among tissues and cell types, potentially contributing to specific sex differences. Here we investigate the role of CTCF, a master chromatin conformation regulator, in regulating escape from XCI. CTCF binding profiles and epigenetic features were systematically examined at constitutive and facultative escape genes using mouse allelic systems to distinguish the inactive X (Xi) and active X (Xa) chromosomes. ResultsWe found that escape genes are located inside domains flanked by convergent arrays of CTCF binding sites, consistent with the formation of loops. In addition, strong and divergent CTCF binding sites often located at the boundaries between escape genes and adjacent neighbors subject to XCI would help insulate domains. Facultative escapees show clear differences in CTCF binding dependent on their XCI status in specific cell types/tissues. Concordantly, deletion but not inversion of a CTCF binding site at the boundary between the facultative escape gene Car5b and its silent neighbor Siah1b resulted in loss of Car5b escape. Reduced CTCF binding and enrichment of a repressive mark over Car5b in cells with a boundary deletion indicated loss of looping and insulation. In mutant lines in which either the Xi-specific compact structure or its H3K27me3 enrichment was disrupted, escape genes showed an increase in gene expression and associated active marks, supporting the roles of the 3D Xi structure and heterochromatic marks in constraining levels of escape. ConclusionOur findings indicate that escape from XCI is modulated both by looping and insulation of chromatin via convergent arrays of CTCF binding sites and by compaction and epigenetic features of the surrounding heterochromatin.

genomics↗

bioRxiv2023

Integrin beta4 promotes DNA damage drug resistance in triple-negative breast cancer via TNFAIP2/IQGAP1/Rac1

Anti-tumor drug resistance is a challenge for human triple-negative breast cancer treatment. Our previous work demonstrated that TNFAIP2 activates RAC1 to promote triple-negative breast cancer cell proliferation and migration. However, the mechanism by which TNFAIP2 activates RAC1 is unknown. In this study, we found that TNFAIP2 interacts with IQGAP1 and Integrin {beta}4. Integrin {beta}4 activates RAC1 through TNFAIP2 and IQGAP1 and confers DNA damage-related drug resistance in triple-negative breast cancer. These results indicate that the Integrin {beta}4/TNFAIP2/IQGAP1/RAC1 axis provides potential therapeutic targets to overcome DNA damage-related drug resistance in triple-negative breast cancer.

cancer biology↗

bioRxiv2023

Imaging Microglia Surveillance during Sleep-wake Cycles in Freely Behaving Mice

Microglia surveillance manifests itself as dynamic changes in cell morphology and functional remodeling. Whether and how microglia surveillance is coupled to brain state switches during natural sleep-wake cycles remain unclear. To address this question, we used miniature two-photon microscopy (mTPM) to acquire time-lapse high-resolution microglia images of the somatosensory cortex, along with EEG/EMG recordings and behavioral video, in freely-behaving mice. We uncovered fast and robust brain state-dependent changes in microglia surveillance, occurring in parallel with sleep dynamics and early-onset phagocytic microglial contraction during sleep deprivation stress. We also detected local norepinephrine fluctuation occurring in a sleep state-dependent manner. We showed that the locus coeruleus-norepinephrine system, which is crucial to sleep homeostasis, is required for both sleep state-dependent and stress-induced microglial responses and {beta}2-adrenergic receptor signaling plays a significant role in this process. These results provide direct evidence that microglial surveillance is exquisitely tuned to signals and stressors that regulate sleep dynamics and homeostasis so as to adjust its varied roles to complement those of neurons in the brain. In vivo imaging with mTPM in freely behaving animals, as demonstrated here, opens a new avenue for future investigation of microglia dynamics and sleep biology in freely behaving animals.

neuroscience↗

bioRxiv2023

Hypothesis-free phenotype prediction within a genetics-first framework

Cohort-wide sequencing studies have revealed that the largest category of variants is those deemed rare, even for the subset located in coding regions (99% of known coding variants are seen in less than 1% of the population1-3). Our understanding of how rare genetic variants influence disease and organism-level phenotypes has achieved limited progress, partly explained by the intrinsic difficulty in statistically evaluating the biological significance of rare events. Here we show that discoveries can instead be made through a knowledge-based approach using protein domains and ontologies (function and phenotype) that considers all coding variants regardless of allele frequency. We describe an ab initio, genetics-first method making molecular knowledge-based interpretations for exome-wide non-synonymous variants for phenotypes at the organism and cellular level. By using this reverse approach, we identify plausible novel genetic causes for developmental disorders that have eluded other established methods and present novel molecular hypotheses for the causal genetics of 40 phenotypes generated from a direct-to-consumer genotype cohort. This system offers a chance to extract further discovery from genetic data after standard tools have been applied.

genomics↗

bioRxiv2022

A Host-Harbored Metabolic Susceptibility of Coronavirus Enables Broad-Spectrum Targeting

Host-based antivirals could offer broad-spectrum therapeutics and prophylactics against the constantly-mutating viruses including the currently-ravaging coronavirus, yet must target cellular vulnerabilities of viruses without grossly endangering the host. Here we show that the master lipid regulator SREBP1 couples the phospholipid scramblase TMEM41B to constitute a host "metabolism-to-manufacture" cascade that maximizes membrane supplies to support coronaviral genome replication, harboring biosynthetic enzymes including Lipin1 as druggable viral-specific-essential (VSE) host genes. Moreover, pharmacological inhibition of Lipin1, by a moonlight function of the widely-prescribed beta-blocker Propranolol, metabolically uncouples the SREBP1-TMEM41B cascade and consequently exhibits broad-spectrum antiviral effects against coronaviruses, Zika virus, and Dengue virus. The data implicate a metabolism-based antiviral strategy that is well tolerated by the host, and a potential broad-spectrum medication against current and future coronavirus diseases.

cell biology↗

bioRxiv2022

Discovery of an alternate mechanism for the inhibition of particulate guanylyl cyclases by the natriuretic peptide 'clearance' receptor

The cardiac natriuretic peptides (NPs) control pivotal physiological actions such as fluid and electrolyte balance, cardiovascular homeostasis, and adipose tissue metabolism by activating their receptor enzymes (NPRA and NPRB). These receptors are homodimers that generate intracellular cyclic guanosine monophosphate (cGMP). The NP receptor NPRC, nicknamed the clearance receptor, lacks a guanylyl cyclase domain; instead, it can bind the NPs to internalize and degrade them. The conventional paradigm is that by competing for and internalizing NPs, NPRC blunts the ability of NPs to signal through NPRA and NPRB. Here we show another previously unknown mechanism by which NPRC can interfere with the cGMP signaling function of the NP receptors. By forming a heterodimer with monomeric NPRA or NPRB, NPRC can prevent the formation of a functional guanylyl cyclase domain and thereby suppress cGMP production in a cell-autonomous manner. Significance StatementNatriuretic peptides (NP) are hormones that are established regulators of vascular and cardiac function, in part through their regulation of fluid and electrolyte balance. NPs signal through particulate guanylyl cyclases (NPRA and NPRB), which are homodimeric membrane-bound receptor enzymes that generate cGMP upon NP binding. Additionally, a silent NP receptor (NPRC) lacks the guanylyl cyclase domain and is a negative regulator of NP signaling. It has been demonstrated that NPRC undergoes internalization and recycling and thus removes NPs, thereby blunting activation of the guanylyl cyclase-containing receptors. Here we show an additional mechanism by which NPRC inhibits NP signaling. Our results show that NPRC can directly interact with NPRA and NPRB, forming non-functional receptor heterodimers with NPRA and NPRB, thereby abrogating NP-evoked cGMP production. This finding establishes another novel mechanistic role for NPRC.

biochemistry↗

bioRxiv2022

Hepatic inactivation of murine Surf4 results in marked reduction in plasma cholesterol

PCSK9 negatively regulates low-density lipoprotein receptor (LDLR) abundance on the cell surface, leading to decreased hepatic clearance of LDL particles and increased levels of plasma cholesterol. We previously identified SURF4 as a cargo receptor that facilitates PCSK9 secretion in HEK293T cells (Emmer et al., 2018). Here, we generated hepatic SURF4-deficient mice (Surf4fl/fl Alb-Cre+) to investigate the physiologic role of SURF4 in vivo. Surf4fl/fl Alb-Cre+ mice exhibited normal viability, gross development, and fertility. Plasma PCSK9 levels were reduced by {bsim}60% in Surf4fl/fl Alb-Cre+ mice, with a corresponding {bsim}50% increase in steady state LDLR protein abundance in the liver, consistent with SURF4 functioning as a cargo receptor for PCSK9. Surprisingly, these mice exhibited a marked reduction in plasma cholesterol and triglyceride levels out of proportion to the partial increase in hepatic LDLR abundance. Detailed characterization of lipoprotein metabolism in these mice instead revealed a severe defect in hepatic lipoprotein secretion, consistent with prior reports of SURF4 also promoting the secretion of apolipoprotein B. Despite a small increase in liver mass and lipid content, histologic evaluation revealed no evidence of steatohepatitis or fibrosis in Surf4fl/fl Alb-Cre+ mice. Acute depletion of hepatic SURF4 by CRISPR/Cas9 or liver-targeted siRNA in adult mice confirms these findings. Together, these data support the physiologic significance of SURF4 in the hepatic secretion of PCSK9 and APOB-containing lipoproteins and its potential as a therapeutic target in atherosclerotic cardiovascular diseases.

physiology↗

bioRxiv2022

The mechanism of DNA sensor cGAS-STING regulating autophagy-related signal pathways

Cyclic GMP-AMP synthase (cGAS) serves as a DNA sensor for recognizing and binding microbial or self-DNA molecules in cells. Upon binding DNA, cGAS produces the second messenger cGAMP to activate the stimulator of interferon genes (STING), which mediates the expression of type 1 interferons (IFN-I) and other cytokines. cGAS-STING-mediated signal pathway plays an important role in innate immune reaction against microbial infections as well as autoimmunity, tumor immunology, and cellular senescence. During this process, cGAS regulates DNA damage repair and induces STING-mediated NF-{kappa}B and MAPK signal pathways in autophagy and lysosome-dependent cell apoptosis. However, the molecular mechanisms of cGAS-STING-mediated autophagy still need to be explored. Here, we found that cGAS-STING promotes autophagy by influencing multiple autophagy-related signal pathways: First, cGAS-STING promotes autophagy by inhibiting the mTOR signaling pathway; Second, cGAS-STING affects autophagy by regulating autophagy-related proteins that are also involved in cGAS-STING-mediated IFN-I and NF-{kappa}B signaling pathways; Third, the Bcl2 can interact with cGAS and STING to induce cGAS-STING-mediated up-regulation of IFN-I signaling pathway and down-regulation of NF-{kappa}B. In addition, we also found that USP19 can significantly reduce the K11-linked ubiquitination of STING in the process of autophagy. Our findings unveiled the functional role and the mechanism of cGAS-STING signaling in regulating autophagy.

molecular biology↗

bioRxiv2022

Assessment of genetic diversity and SNP marker development within peanut germplasm in Taiwan by RAD-seq

The cultivated peanut (Arachis hypogaea L.) is an important oil crop but has a narrow genetic diversity. Molecular markers can be used to probe the genetic diversity of various germplasm. In this study, the restriction site associated DNA (RAD) approach was utilized to sequence 31 accessions of Taiwanese peanut germplasm, leading to the identification of a total of 17,610 single nucleotide polymorphisms (SNPs). The origin of these 31 accessions is contrasted so the global subset (n = 17) has greater genetic diversity than the local subset (n = 14). Concerning botanical varieties, the var. fastigiata subset has greater genetic diversity than the other two subsets of var. vulgaris and var. hypogaea, suggesting that novel genetic resources should be introduced into breeding programs to enhance genetic diversity. Principal component analysis (PCA) using genotyping data separated the 31 accessions into three clusters largely according to the botanical varieties, consistent with the PCA result for 282 accessions genotyped by 14 kompetitive allele-specific PCR (KASP) markers developed in this study. The SNP markers identified in this work not only revealed the genetic relationship and population structure of current germplasm in Taiwan, but also offer an efficient tool for breeding and further genetic applications.

genetics↗

bioRxiv2022

ABIOTIC STRESS GENE 1 mediates aroma volatiles accumulation by activating MdLOX1a in apple

Fruit aroma is an important organoleptic quality, which influences consumer preference and market competitiveness. Aroma compound synthesis pathways in plants have been widely identified of which the lipoxygenase pathway is crucial for fatty acid catabolism to form esters in apple. However, the regulatory mechanism of this pathway remains elusive. In this study, linear regression analysis and transgene verification revealed that the lipoxygenase MdLOX1a participates in ester biosynthesis. Yeast one-hybrid library screening indicated that a novel abiotic stress gene, MdASG1 (ABIOTIC STRESS GENE 1), was a positive regulator of the MdLOX1a promoter and ester production based on yeast one-hybrid and dual-luciferase assays, and correlation analysis among eight apple cultivars. Overexpression of MdASG1 in apple and tomato stimulated the lipoxygenase pathway and increased the fatty acid-derived volatile content, whereas the latter was decreased by MdASG1 silencing. Furthermore, MdASG1 overexpression enhanced the salt-stress tolerance of tomato and apple Orin calli accompanied by a higher content of fatty acid-derived volatiles compared with that of non-stressed transgenic tomato fruit. Collectively, these findings indicate that MdASG1 activates MdLOX1a expression and participates in the lipoxygenase pathway, subsequently increasing the accumulation of aroma compounds especially under moderate salt stress treatment. The results also provide insight into the regulation of aroma production, and the potential strategy of prudent development and utilization of saline-alkali land to produce high-quality fruit, thereby reducing pressure on arable land and ensuring national food security. One-sentence SummaryMdASG1 directly activates MdLOX1a expression to promote aroma volatiles accumulation especially under moderate salt stress.

molecular biology↗

bioRxiv2022

Evolution of inflammation and immunity in a dengue virus 1 human infection model

Dengue virus (DENV) infections are significant sources of morbidity and mortality throughout the tropics and subtropics. Over 400 million infections are estimated to occur every year, resulting in nearly 100 million symptomatic infections and over 20,000 deaths. Early immune response kinetics to infection remain unclear, in large part due to the variable incubation period exhibited by the DENVs after introduction into a susceptible host. To fill this knowledge gap, we performed a comprehensive virologic and immunologic analysis of individuals experimentally infected with the under-attenuated DENV-1 strain 45AZ5. This analysis captured both the kinetics and composition of the innate, humoral, and cellular immune responses elicited by experimental DENV-1 infection, as well as virologic and clinical features. Revealed in this analysis was a robust DENV-specific IgA antibody response that manifested between the appearance of DENV-specific IgM and IgG in all challenged individuals, as well as the presence of a non-neutralizing/NS1-specific antibody response that was delayed relative to the appearance of DENV-virion specific humoral immunity. RNAseq analysis also revealed several distinct and temporally-restricted gene modules that allowed for the identification and differentiation of the innate and adaptive immune response to DENV-infection. Our analysis provides a detailed description, in time and space, of the evolving matrix of DENV-elicited human inflammation and immunity and reveals several previously unappreciated immunological aspects of primary DENV-1 infection that can inform countermeasure development and evaluation.

immunology↗

bioRxiv2022

XPD protects CTCF-Cohesin binding sites from somatic mutagenesis

Xeroderma pigmentosum group D (XPD) is a DNA helicase with critical functions in transcription initiation and nucleotide excision repair. Missense mutations in XPD are putative drivers in around 10% of bladder cancers (BLCA), but the associated mutational process remains poorly understood. Here, we examine the somatic mutational landscape of XPD wild-type (n=343) and mutant (n=39) BLCA whole genomes. The genome-wide distribution of somatic mutations is significantly altered in XPD mutants, affecting both APOBEC and non-APOBEC associated mutational processes. Specifically, XPD mutants are enriched in T[C>T]N mutations (SBS2) with altered correlation with replication timing. At a locoregional genomic level, mutant XPD BLCA had striking T>G mutation hotspots at CTCF-cohesin binding sites (CBS) with evidence linking XPD to genomic uracil repair. Leveraging differential distribution of somatic mutations, we developed a machine-learning model for predicting pathogenic XPD mutations, which we validated in an independent TCGA cohort with 100% accuracy. Our model enabled the discovery of missed XPD mutation calls and uncovered pathogenic non-hotspot XPD mutations in bladder cancer. Our study reveals how XPD mutations redistribute somatic mutations in cancer genomes and provides a genome sequencing approach to differentiate driver and passenger XPD mutations.

molecular biology↗

bioRxiv2021

Gasotransmitter H2S accelerates seed germination via activating AOX mediated cyanide-resistant respiration pathway

Hydrogen sulfide (H2S) has been witnessed as a crucial gasotransmitter involving in various physiological processes in plants. H2S signaling has been reported to involve in regulating seed germination, but the underlying mechanism remains poorly understood. Here, we found that endogenous H2S production was activated in germinating Arabidopsis seeds, correlating with upregulated both the transcription and the activity of enzymes (LCD and DES1) responsible for H2S production. Moreover, NaHS (the H2S donor) fumigation significantly accelerated seed germination, while H2S-generation defective (lcd/des1) seeds exhibited decreased germination speed. Further results indicated that the alternative oxidase (AOX), a cyanide-insensitive terminal oxidase, can be stimulated by imbibition, and the expression of AOX genes was provoked lag behind H2S production during germination. Additionally, exogenous H2S fumigation significantly reinforced imbibition induced enhancement of AOX1A expression, and mediated post-translational modification to keep AOX in its reduced and active state, which mainly involved H2S induced increase of the GSH/GSSG ratio and the cell reducing power. Consequently, H2S signaling acts as a trigger to induce AOX mediated cyanide-resistant respiration to accelerate seed germination. Our study correlates H2S signaling to cyanide metabolism, which also participates in endogenous H2S generation, providing evidence for more extensive studies of H2S signaling. HighlightGasotransmitter H2S provokes AOX mediated cyanide-resistant respiration, mainly through both long-term (up-regulating AOX1A expression) and short-term (inducing post-translational activation of AOX) regulatory modes, to accelerate seed germination.

molecular biology↗

bioRxiv2021

Monomeric IgA antagonizes IgG-mediated enhancement of DENV infection

Dengue virus (DENV) is a prevalent human pathogen, infecting approximately 400 million individuals per year and causing symptomatic disease in approximately 100 million. A distinct feature of dengue is the increased risk for severe disease in some individuals with preexisting DENV-specific immunity. One proposed mechanism for this phenomenon is antibody-dependent enhancement (ADE), in which poorly-neutralizing IgG antibodies from a prior infection opsonize DENV to increase infection of Fc gamma receptor-bearing cells. While IgM and IgG are the most commonly studied DENV-reactive antibody isotypes, our group and others have described the induction of DENV-specific serum IgA responses during dengue. We hypothesized that monomeric IgA would be able to neutralize DENV without the possibility of ADE. To test this, we synthesized IgG and IgA versions of two different DENV-reactive monoclonal antibodies. We demonstrate that isotype-switching does not affect the antigen binding and neutralization properties of the two mAbs. We show that DENV-reactive IgG, but not IgA, mediates ADE in an Fc gamma receptor-positive K562 cells. Furthermore, we show that IgA potently antagonizes the ADE activity of IgG. These results suggest that levels of serum DENV-reactive IgA induced by DENV infection might regulate the overall ADE activity of DENV-immune plasma in vivo and warrants further study as a predictor of disease risk and/or therapeutic.

immunology↗

bioRxiv2021

The Proteomic Architecture of Schizophrenia Cerebral Organoids Reveals Alterations in GWAS and Neuronal Development Factors

Schizophrenia (Scz) is a brain disorder that has a typical onset in early adulthood but otherwise maintains unknown disease origins. Unfortunately, little progress has been made in understanding the molecular mechanisms underlying neurodevelopment of Scz due to ethical and technical limitations in accessing developing human brain tissue. To overcome this challenge, we have previously utilized patient-derived Induced Pluripotent Stem Cells (iPSCs) to generate self-developing, self-maturating, and self-organizing 3D brain-like tissue known as cerebral organoids. As a continuation of this prior work [1], here we provide a molecular architectural map of the developing Scz organoid proteome. Utilizing iPSCs from n = 25 human donors (n = 8 healthy Ctrl donors, and n = 17 Scz patients), we generated 3D human cerebral organoids, employed 16-plex isobaric sample-barcoding chemistry, and simultaneously subjected samples to comprehensive high-throughput liquid-chromatography/mass-spectrometry (LC/MS) quantitative proteomics. Of 3,705 proteins identified by high-throughput proteomic profiling, we identified that just ~2.62% of the organoid global proteomic landscape was differentially regulated in Scz organoids. In sum, just 43 proteins were up-regulated and 54 were down-regulated in Scz patient-derived organoids. Notably, a range of neuronal factors were depleted in Scz organoids (e.g., MAP2, TUBB3, SV2A, GAP43, CRABP1, NCAM1 etc.). Based on global enrichment analysis, alterations in key pathways that regulate nervous system development (e.g., axonogenesis, axon development, axon guidance, morphogenesis pathways regulating neuronal differentiation, as well as substantia nigra development) were perturbed in Scz patient-derived organoids. We also identified prominent alterations in two novel GWAS factors, Pleiotrophin (PTN) and Podocalyxin (PODXL), in Scz organoids. In sum, this work serves as both a report and a resource whereby researchers can leverage human-derived neurodevelopmental data from Scz patients, which can be used to mine, compare, contrast, or orthogonally validate novel factors and pathways related to Scz risk identified in datasets from observational clinical studies and other model systems.

neuroscience↗

bioRxiv2021

Variable Induction of Pro-inflammatory Cytokines by Commercial SARS CoV-2 Spike Protein Reagents: Potential Impacts of LPS on In Vitro Modeling and Pathogenic Mechanisms In Vivo

Proinflammatory cytokine production following infection with severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) is associated with poor clinical outcomes. Like SARS CoV-1, SARS CoV-2 enters host cells via its spike protein, which attaches to angiotensin-converting enzyme 2 (ACE2). As SARS CoV-1 spike protein is reported to induce cytokine production, we hypothesized that this pathway could be a shared mechanism underlying pathogenic immune responses. We herein compared the capabilities of Middle East Respiratory Syndrome (MERS), SARS CoV-1 and SARS CoV-2 spike proteins to induce cytokine expression in human peripheral blood mononuclear cells (PBMC). We observed that only specific commercial lots of SARS CoV-2 induce cytokine production. Surprisingly, recombinant SARS CoV-2 spike proteins from different vendors and batches exhibited different patterns of cytokine induction, and these activities were not inhibited by blockade of spike protein-ACE2 binding using either soluble ACE2 or neutralizing anti-S1 antibody. Moreover, commercial spike protein reagents contained varying levels of endotoxin, which correlated directly with their abilities to induce cytokine production. The lipopolysaccharide (LPS) inhibitor, polymyxin B, blocked this cytokine induction activity. In addition, SARS CoV-2 spike protein avidly bound soluble LPS in vitro, rendering it a cytokine inducer. These results not only suggest caution in monitoring the purity of SARS CoV-2 spike protein reagents, but they indicate the possibility that interactions of SARS CoV-2 spike protein with LPS from commensal bacteria in virally infected mucosal tissues could promote pathogenic inflammatory cytokine production.

microbiology↗

bioRxiv2021

Genome recombination-mediated tRNA up-regulation conducts general antibiotic resistance of bacteria at early stage

Bacterial antibiotic resistance sets a great challenge to human health. It seems that the bacteria can spontaneously evolve resistance against any antibiotic within short time without the horizontal transfer of heterologous genes and before accumulating drug-resistant mutations. We have shown that the tRNA-mediated translational regulation counteracts the reactive oxygen species in bacteria. In this study, we demonstrated that isolated and subcultured Escherichia coli elevated its tRNAs under antibiotic stress to rapidly provide antibiotic resistance, especially at the early stage, before upregulating the efflux pump and evolving resistance mutations. The DNA recombination system repaired the antibiotic-induced DNA breakage in the genome, causing numerous structural variations. These structural variations are overrepresented near the tRNA genes, which indicated the cause of tRNA up-regulation. The strains knocking out the recombination system could not up-regulate tRNAs, and coincidently, they could hardly evolve antibiotic resistance in multiple antibiotics, respectively. With these results, we proposed a multi-stage model of bacterial antibiotic resistance in an isolated scenario: the early stage (recombination - tRNA up-regulation - translational regulation); the medium stage (up-regulation of efflux pump); the late stage (resistance mutations). These results also indicated that the bacterial DNA recombination system and tRNA could be targeted to retard the bacterial spontaneous drug resistance.

microbiology↗

bioRxiv2021

High-throughput tandem-microwell assay for ammonia repositions FDA-Approved drugs to Helicobacter pylori infection

To date, little attempt has been made to develop new treatments for Helicobacter pylori (H. pylori), although the community is aware of the shortage of treatments for H. pylori. In this study, we developed a 192-tandem-microwell-based high-throughput-assay for ammonia that is a known virulence factor of H. pylori and a product of urease. We could identify few drugs, i.e. panobinostat, dacinostat, ebselen, captan and disulfiram, to potently inhibit the activity of ureases from bacterial or plant species. These inhibitors suppress the activity of urease via substrate-competitive or covalent-allosteric mechanism, but all except captan prevent the antibiotic-resistant H. pylori strain from infecting human gastric cells, with a more pronounced effect than acetohydroxamic acid, a well-known urease inhibitor and clinically used drug for the treatment of bacterial infection. This study offers several bases for the development of new treatments for urease-containing pathogens and to study the mechanism responsible for the regulation of urease activity.

biochemistry↗